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DOI: 10.1186/s13023-015-0238-5

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Citation for published version (APA): Thoenes, M., Zimmermann, U., Ebermann, I., Ptok, M., Lewis, M. A., Thiele, H., Morlot, S., Hess, M. M., Gal, A., Eisenberger, T., Bergmann, C., Nürnberg, G., Nürnberg, P., Steel, K. P., Knipper, M., & Bolz, H. J. (2015). OSBPL2 encodes a protein of inner and outer hair cell stereocilia and is mutated in autosomal dominant hearing loss (DFNA67). Orphanet Journal of Rare Diseases, 10(1), [15]. https://doi.org/10.1186/s13023-015-0238-5

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Download date: 01. Oct. 2021 Thoenes et al. Orphanet Journal of Rare Diseases (2015) 10:15 DOI 10.1186/s13023-015-0238-5

RESEARCH Open Access OSBPL2 encodes a protein of inner and outer hair cell stereocilia and is mutated in autosomal dominant hearing loss (DFNA67) Michaela Thoenes1, Ulrike Zimmermann2, Inga Ebermann1, Martin Ptok3, Morag A Lewis4, Holger Thiele5, Susanne Morlot6, Markus M Hess7, Andreas Gal8, Tobias Eisenberger9, Carsten Bergmann9,10, Gudrun Nürnberg5, Peter Nürnberg5,11, Karen P Steel4, Marlies Knipper2 and Hanno Jörn Bolz1,9*

Abstract Background: Early-onset hearing loss is mostly of genetic origin. The complexity of the hearing process is reflected by its extensive genetic heterogeneity, with probably many causative genes remaining to be identified. Here, we aimed at identifying the genetic basis for autosomal dominant non-syndromic hearing loss (ADNSHL) in a large German family. Methods: A panel of 66 known deafness genes was analyzed for mutations by next-generation sequencing (NGS) in the index patient. We then conducted genome-wide linkage analysis, and whole-exome sequencing was carried out with samples of two patients. Expression of Osbpl2 in the mouse cochlea was determined by immunohistochemistry. Because Osbpl2 has been proposed as a target of miR-96, we investigated homozygous Mir96 mutant mice for its upregulation. Results: Onset of hearing loss in the investigated ADNSHL family is in childhood, initially affecting the high frequencies and progressing to profound deafness in adulthood. However, there is considerable intrafamilial variability. We mapped a novel ADNSHL locus, DFNA67, to chromosome 20q13.2-q13.33, and subsequently identified a co-segregating heterozygous frameshift mutation, c.141_142delTG (p.Arg50Alafs*103), in OSBPL2, encoding a protein known to interact with the DFNA1 protein, DIAPH1. In mice, Osbpl2 was prominently expressed in stereocilia of cochlear outer and inner hair cells. We found no significant Osbpl2 upregulation at the mRNA level in homozygous Mir96 mutant mice. Conclusion: The function of OSBPL2 in the hearing process remains to be determined. Our study and the recent description of another frameshift mutation in a Chinese ADNSHL family identify OSBPL2 as a novel gene for progressive deafness. Keywords: OSBPL2, DFNA67, Autosomal dominant hearing loss

Background NSHL). Approximately 20% of patients have autosomal Hearing impairment is the most common sensory dis- dominantly-inherited forms (ADNSHL) and typically order, affecting approximately 1/500 newborns. In devel- display postlingual progressive hearing impairment. oped countries, most cases are of genetic origin, and Sixty-five ADNSHL loci have been officially designated, there is extensive allelic and non-allelic heterogeneity. and 30 causative genes have been reported [1-3]. Because In 70% of hearing-impaired neonates, the sensory def- of the extensive genetic heterogeneity of hearing impair- icit is non-syndromic (non-syndromic hearing loss, ment, the identification of the causative mutation in single patients has been the exception until recently. With the advent of next-generation sequencing (NGS), deafness * Correspondence: [email protected] 1Institute of Human Genetics, University Hospital of Cologne, Cologne, genes have become accessible to comprehensive genetic Germany analysis and routine genetic testing by targeted NGS of 9 Center for Human Genetics, Bioscientia, Ingelheim, Germany “gene panels” [4]. Full list of author information is available at the end of the article

© 2015 Thoenes et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Thoenes et al. Orphanet Journal of Rare Diseases (2015) 10:15 Page 2 of 10

Methods SAMtools [8], Picard (http://broadinstitute.github.io/picard/) Patients and GATK [9]. Variants were filtered against dbNSFP v2.0 Samples of the German family reported herein (Figure 1) [10], dbSNP v137, the Human Gene Mutation Database were obtained with written informed consent. Clinical (HGMD® Professional 2013.2) [11] and our in-house data- investigations were conducted according to the Declar- base. The cutoff for the maximum minor allele frequency ation of Helsinki, and the study was approved by the (MAF) was set to 1% [12]. Nonsense, frameshift and institutional review board of the Ethics Committee of canonical splice site variants were regarded likely patho- the University Hospital of Cologne. The affected subjects genic. SNVs were assessed using SIFT [13], Mutation underwent detailed audiological evaluations (e.g., pure Taster [14], PolyPhen-2 [15], AlignGVGD [16,17], Pmut tone audiometric air conduction, bone conduction, speech [18], NNSPLICE v0.9 [19] and NetGene2 [20,21]. SeqPilot reception threshold, otoacoustic emissions, and imped- SeqNext module (v4.0.1, JSI medical systems) was used ance audiometry, phoneme discrimination), except IV:12 for visualization and final assessment of SNVs. who reports intermittent hearing impairment (related to stress). Following the recommendations of the EU HEAR project [5], hearing loss was classified as mild Linkage analysis and locus designation (20 – 40 dB), moderate (41 – 70 dB), severe (71 – 95 dB), DNA samples from seven affected and six unaffected or profound (>95 dB). This classification, however, was members of the family (Figure 1) were genotyped using difficult in some cases, because at least two patients the Affymetrix GeneChip Human Mapping 10 K SNP (V:2, V:3) had near normal hearing in the 250 – 1000 Hz array Xba142. The gender was verified by counting het- region, but a loss of about 70 – 90 dB in the higher erozygous single nucleotide polymorphisms (SNPs) on frequencies. the X chromosome. Relationship errors were evaluated with the help of the program Graphical Representation of GJB2 analysis and targeted NGS of a deafness gene panel Relationships [22]. Linkage analysis was performed assum- The GJB2 gene was directly sequenced in the index ing autosomal dominant inheritance, full penetrance, patient, IV:10. His sample was then subjected to NGS and a disease gene frequency of 0.0001. Multipoint LOD for 66 genes (1,259 coding exons) that have been associ- scores were calculated using the program ALLEGRO [23]. ated with NSHL and selected forms of SHL on a MiSeq Haplotypes were reconstructed with ALLEGRO and system (Illumina) as described previously [6]. In brief, presented graphically with HaploPainter [24]. All data sheared DNA was ligated to bar-coded adaptors for handling was performed using the graphical user interface multiplexing. Exons were targeted by an in-solution ALOHOMORA [25]. customized sequence capture library (NimbleGen). Amp- Following the identification of the causative mutation in lified enriched DNA was directly subjected to NGS OSBPL2, DFNA67 was assigned as a novel locus designa- (MiSeq). Reads were mapped against the hg19 human tion for ADNSHL by the Human Gene Nomenclature reference genome using BWA [7] and processed with Committee, HGNC.

Figure 1 Pedigree and genotypes of the DFNA67 family. M, OSBPL2 mutation; WT, wildtype. Blue stars, individuals whose samples were subjected to genome-wide SNP genotyping for linkage analysis. Green stars, individuals whose samples were subjected to WES. Thoenes et al. Orphanet Journal of Rare Diseases (2015) 10:15 Page 3 of 10

Whole-exome sequencing denaturation phase of 94°C for 4 min, 35 cycles of 30 s Samples of two affected family members, IV:10 and V:3, denaturation (94°C), 30 s annealing (58°C), 30 s extension were subjected to whole-exome sequencing (WES). (72°C), and a final extension phase of 5 min at 72°C. The DNA (1 μg) was fragmented using sonication technology PCR products were analyzed on ethidium bromide agar- (Covaris). Fragments were enriched using the SeqCap ose gels. Fragments were extracted using QIAquick Gel EZ Human Exome Library v2.0 kit (Roche NimbleGen) Extraction Kit (Qiagen), cloned and sequenced. and subsequently sequenced on an Illumina HiSeq 2000 sequencing instrument using a paired-end 2 × 100-bp Tissue preparation and immunohistochemistry protocol. This generated 6.1 and 8.2 Gb of mapped se- Cochleae from adult mice (aged between P20 and six quences with a mean coverage of 80-fold and 101-fold, a months) were used to prepare cryosections for immuno- 30-fold coverage of 89% and 89% of the target sequences fluorescence microscopy. For immunohistochemistry on and a 10-fold coverage of 96% and 95% of the target se- cryosections, the temporal bone of mature mice was dis- quences. For data analysis, the Varbank pipeline (v.2.3) sected on ice and immediately fixed using Zamboni’s and filter interface was used (https://varbank.ccg.uni- fixative [29] containing picric acid by infusion through koeln.de). Primary data were filtered according to signal the round and oval window and incubated for 15 min on purity by Illumina Real-Time Analysis (RTA) software ice, followed by rinsing with phosphate-buffered saline (v1.8). Subsequently, the reads were mapped to the hu- (PBS) and decalcification in rapid bone decalcifier man genome reference build hg19 using the bwa-aln (Eurobio, Fisher-Scientific). After injection of 25% su- alignment algorithm. GATK v1.6 [9] was used to mark crose in PBS, pH 7.4, cochleae were embedded in O.C.T. duplicated reads, perform local realignment around compound (Miles Laboratories, Elkhart, IN). Tissues short insertions and deletions, recalibrate the base qual- were then cryosectioned at 10 μm thickness, mounted ity scores, and call SNPs and short indels. Scripts devel- on SuperFrost*/plus microscope slides, dried for 1 h and oped in-house at the CCG were applied to variants stored at -20°C before use. Cryosections were thawed and predicted to result in protein changes, variants affecting permeabilized with 0.1% Triton X-100 (Sigma Aldrich) for donor and acceptor splice sites, and those overlapping 3 min at room temperature, blocked with 1% BSA in PBS, with known variants. Acceptor and donor splice site mu- and incubated with primary antibody in 0.5% BSA in PBS tations were analyzed with a maximum-entropy model overnight at +4°C. For double labeling studies, specimens and filtered for their putative effect on splicing. In particu- were simultaneously incubated with both antibodies for lar, we focused on the linkage regions (chr12:49,329,157- identical time periods. As primary antibodies we used: 52,752,362 and chr20:52,882,032-61,366,354; hg19) and anti-ORP-2 (OSBPL2) antibody (goat, Santa Cruz Biotech- filtered for high-quality (>15-fold coverage; quality > 25; nology, Inc., sc-66570, dilution 1:50), anti-prestin antibody VQSR > -2), and for rare (MAF <0.005) and heterozygous (rabbit [30], dilution 1:5000), and anti-otoferlin antibody variants. Several genome-wide databases (dbSNP Build (rabbit [31], dilution 1:10000). Primary antibodies were 135, 1000 Genomes Project database build 20110521 [26] detected with Cy3- (Jackson Immunoresearch Laborator- and the public Exome Variant Server, NHLBI, Seattle, ies) and Alexa488- (Invitrogen, Life Technologies GmbH) build ESP6500 [27]) were checked for the presence of the conjugated secondary antibodies. Sections embedded with identified variants. To exclude pipeline-related artifacts Vectashield mounting medium containing nuclear marker (MAF <0.005), we also filtered against an in-house data- DAPI (Vector Laboratories). Sections were viewed using base containing variants from 511 exomes of individuals an Olympus BX61 microscope equipped with motorized with epilepsy. Finally, we filtered for variants present in z-axis and epifluorescence illumination. To increase dis- both patients. Verification of sequence variants and segre- play resolution, cochlear slices were imaged over a dis- gation analyses were carried out by Sanger sequencing. tance of several micrometers (30 × 0,27 μm, ~8 μm) in an Sequence data for OSBPL2 (MIM 606731) were compared image stack along the z-axis (z-stack), followed by 3- to the reference sequence NM_144498.2. dimensional deconvolution using cellSens Dimension module with the advanced maximum likelihood estima- RT-PCR from cochlear tissue tion algorithm (ADVMLE; OSIS). Images were acquired mRNA from mouse postnatal day 19 (P19) and rat P17 using a CCD camera and analyzed with cellSens software cochleae was isolated as described earlier [28]. A 298 bp (Olympus Soft Imaging Solutions, OSIS). Images were fragment of Osbpl2 was amplified using the following processed with Photoshop. primer sequences: 5′-CCAACTCTGCTCAGATGTACA AC-3′ (forward) and 5′-GCTGTACGCCGGCCATTAC Comparison of Osbpl2 mRNA levels between wildtype and TTTGA-3′ (reverse). PCR was performed with PuRe- homozygous Mir96 mutant diminuendo (Mir96Dmdo)mice Taq TM Ready-To-GoTM PCR beads (GE Healthcare). Organ of Corti dissection, RNA extraction and cDNA The amplification conditions consisted of an initial creation were carried out as described previously [32]. Thoenes et al. Orphanet Journal of Rare Diseases (2015) 10:15 Page 4 of 10

Primers were purchased from Applied Biosystems (Hprt1: has been annotated in dbSNP (rs199589052), but no MAF Mm01318747_g1, Jag1: Mm01270190_m1, Osbpl2:Mm01 is available. Biallelic mutations in SLC11A2 cause auto- 210488_m1). Hprt1 was used as an internal control, and somal recessive hypochromic microcytic anemia with iron Jag1, which is expressed in supporting cells [32-34], was overload but there is no mention of any hearing impair- used to control for the quantity of sensory tissue present. ment [35]. It is therefore unlikely that heterozygous Pairs were only used when Jag1 levels did not differ sig- SLC11A2 mutations cause ADNSHL. A missense variant, nificantly between wildtype and homozygote littermates c.53G>A (p.Arg18His), was identified in CELA1 (chymo- (p > 0.05). Three animals were genotyped and at least trypsin-like elastase family, member 1). Because of its three technical replicates were performed on each pair. expression in skin tissue, CELA1 had been considered a The reaction was run on a CFX Connect machine using candidate for skin disease. However, a common frameshift Bio-Rad SsoFast and SsoAdvanced Master mixes (Bio-Rad polymorphism questioned essentiality of this gene [36]. Laboratories, cat. nos. 1725232, 1725281). Moreover, the p.Arg18His variant has an MAF of 0.28% which is not compatible with a mutation causing a rare Results autosomal dominant disorder. Clinical characterization of DFNA67 patients There were two heterozygous missense variants in In most affected family members, bilateral sensorineural genes contained in the candidate region on chromosome non-syndromic hearing loss was first noted in the early 20: The variant c.1202G>C (p.Arg401Pro) in GTPBP5 second decade of life and affected initially the high fre- affects an evolutionarily non-conserved residue and has quencies. However, the age of onset varied between 10 been annotated as a polymorphism, rs200118420, with (patients IV:7, V:6) and 30 years of age (III:9). Real onset an MAF of 0.04%. A variant in DIDO1 (c.1738A>C; p. may have been earlier in many cases: Retrospectively, Thr580Pro), also affects a non-conserved residue. Mutant the parents of patient V:3 assume that hearing was Dido -/- and +/- mice appeared grossly normal. With already impaired around the age of 2 years. Progression time, some heterozygous mice showed abnormalities in of hearing loss was also widely variable. It was mild in spleen, bone marrow, and peripheral blood, overlapping younger individuals but severe to profound at later with symptoms of myeloid dysplasia or myeloid prolifera- stages (Figure 2) and required cochlear implantation be- tion [37]. Taken together, none of these three missense tween 27 and 50 years of age in five family members. On variants appeared to be a promising candidate for the the mild end of the spectrum, patient IV:10 noted onset ADNSHL-causing mutation. of hearing loss at 22 years of age and started using hear- A nonsense mutation, c.287C>G (p.Ser96*), was found ing aids at 34 years. No audiological data were available in both patients in SLC17A9, a gene directly adjacent for IV:12, a 39-years old carrier of the OSBPL2 mutation, (61,583,999 – 61,599,949) to the telomeric boundary of who does not use hearing aids but claims worse hearing the chromosome 20 locus. According to the Exome Vari- in stressful situations. III:7 and V:2 reported progression ant Server, the SLC17A9 nonsense variant has an MAF of of hearing loss in the course of pregnancy and birth of 0.06%, and compatible with the gene’s localization just children (see Table 1 for a summary of clinical data). outside the candidate locus, it was carried by three healthy Vestibular symptoms were not reported. individuals of the family (II:5, III:10, IV:13). Moreover, p. Ser96*SLC17A9 was also present in heterozygous state in Mapping of chromosomal candidate loci three samples of our in-house database: A patient with Using ALLEGRO we identifed two genomic regions epilepsy, a patient with amyotrophic lateral sclerosis and withamaximumLODscoreof2.7.TheobtainedLOD the healthy mother of that patient. None of these three score was the maximum possible LOD score in this individuals had hearing loss. family. We found linkage to a 3.4 Mb interval between We identified only one truncating variant in a gene SNPs rs1316607 and rs725029 on chromosome 12 contained in a mapped candidate region: Both patients (49,329,157 – 52,752,362) and to a 8.4 Mb interval carried the frameshift mutation c.141_142delTG (p. between rs2065042 and rs720607 on chromosome 20 Arg50Alafs*103) in OSBPL2 (Figure 3B,C), a gene from (52,882,032 – 61,366,354) (Figure 3 A). the chromosome 20 region. This variant has not been annotated in any of the above databases, no allele fre- Targeted NGS, WES and segregation analysis of the quency is available, and it co-segregated perfectly with candidate variants identified hearing loss in the family. No mutation was identified in GJB2 and in subsequent targeted NGS of 66 known deafness genes. After stringent Expression of Osbpl2 in the murine cochlea filtering of WES data, only two heterozygous variants We analyzed the expression of Osbpl2 at the transcrip- remained that localized to the chromosome 12 candidate tional level of post-hearing animals using RT-PCR with region: The c.1516C>G (p.Arg506Gly) variant in SLC11A2 mRNA from whole mouse (P19) and rat (P17) cochlea. Thoenes et al. Orphanet Journal of Rare Diseases (2015) 10:15 Page 5 of 10

IV:7 IV:9

37 ys 38 ys

IV:10 V:2

36 ys 15 ys 42 ys 19 ys 23 ys

V:3 V:6

19 ys 12 ys 25 ys 16 ys 18 ys

Figure 2 Exemplary audiograms of patients from the DFNA67 family. Hearing thresholds are shown for the more severely affected side.

The amplification product of Osbpl2 with the appropriate Comparison of Osbpl2 mRNA levels between wildtype size (298 bp) was found in both mouse (Figure 3D, lane 2) and homozygous Mir96 mutant mice and rat cochlea (Figure 3D; lane3). Next, we analyzed No significant difference was observed between wildtype Osbpl2 expression in the organ of Corti of mice at the and homozygote levels of Osbpl2 mRNA in the organ of protein level using anti-Osbpl2 antibody in combination Corti (Additional file 1). with either anti-prestin antibody used as an outer hair cell (OHC) marker or anti-otoferlin antibody used as an inner Discussion hair cell (IHC) marker. Osbpl2 was detected in stereocilia Mutations in approximately 30 genes have been impli- of both OHCs (Figure 4A,B) and IHCs (Figure 4C,D). We cated in ADNSHL (with variable evidence) [2]. The found no difference in expression of Osbpl2 between P20 respective gene products fall into many different cat- and 6-month-old mice (not shown). egories and comprise ion channels and transporters, Thoenes et al. Orphanet Journal of Rare Diseases (2015) 10:15 Page 6 of 10

Table 1 Summary of clinical data Individual Age (ys.) Hearing loss Age of onset (ys.) Course OSBPL2 II:1 36 (†)no no sample II:2 69 (†) not noted by any of the five children, ? ? no sample but nasal pronunciation II:5 93 (†)no wildtype II:7 78 (†)no no sample III:2 76 no wildtype III:4 71 unilateral (untreated otitis media) wildtype III:6 74 (†)no wildtype III:7 71 yes 12 worse after birth of children, p.Arg50Alafs*103 stress; CI around 50 ys. III:8 67 no wildtype III:9 61 yes 30 CI at 50 ys. p.Arg50Alafs*103 III:10 64 only temporary, episodes of acute wildtype hearing loss IV:6 58 no wildtype IV:7 50 yes 10 CI at 36 ys. p.Arg50Alafs*103 IV:8 50 no wildtype IV:9 49 yes 12 CI at 39 ys. p.Arg50Alafs*103 IV:10 45 yes 22 hearing aids at 34 ys. p.Arg50Alafs*103 IV:11 44 no wildtype IV:12 39 no data from investigations; ? ? p.Arg50Alafs*103 hearing worse under stress IV:13 35 no; two healthy daughters no sample V:2 28 yes 15 worsening after birth of children; CI at 27 ys. p.Arg50Alafs*103 V:3 26 yes 11 hearing aids at 12 ys. p.Arg50Alafs*103 V:4 15 no wildtype V:5 16 no wildtype V:6 20 yes 10 hearing aids at 15 ys. p.Arg50Alafs*103 V:7 17 no wildtype VI:1 3 no (normal OAEs at 3 ys.) wildtype VI:2 4 months no motor molecules, components of the extracellular matrix, organelle membranes and assembly of protein complexes, the cytoskeleton, adhesion complexes etc. [1]. thereby impacting signaling, vesicle transport and lipid Of the five heterozygous variants found in genes from metabolism [39]. the mapped candidate regions on chromosomes 12 and Osbpl2 was among 132 mRNAs with 3′UTRs pre- 20 in our family, the OSBPL2 frameshift mutation repre- dicted to contain potential target sites for miR-96 [32], a sented a reasonable candidate for the ADNSHL-causing microRNA whose mutations cause progressive hearing mutation (see results section). OSBPL2 does not belong loss in mice and humans [32,40], but we found no up- to any of the above protein classes. It is part of a 12- regulation of Osbpl2 in Mir96 mutant mice (diminu- member, evolutionarily highly conserved family of lipid endo) (Additional file 1). However, besides its previously binding/transfer proteins, the oxysterol binding proteins reported expression in the mouse organ of Corti at the (OSBPs) and related proteins (OSBPLs) that share the onset of hearing [41], there are several lines of evi- characteristic OSBP signature, EQVSHHPP [38]. OSBPL dence that OSBPL2 isthegeneunderlyingADNSHL proteins play an important role in non-vesicular intracel- in the family described herein: The frameshift mutation lular transport of lipids, particularly oxysterol, a deriva- c.141_142delTG very likely represents a loss-of-function tive of cholesterol. OSBPLs serve as sterol sensors and allele causing OSBPL2 haploinsufficiency. It either results transporters that modulate the lipid composition of cell in a truncated non-functional protein of 151 residues Thoenes et al. Orphanet Journal of Rare Diseases (2015) 10:15 Page 7 of 10

cM B

C G G A A A A C T G G G G A G A G G G A G A G G

1 2 3 4 5 6 10 14

OSBPL2: c.141_142delTG (p.Arg50Alafs*103) D

Figure 3 (See legend on next page.) Thoenes et al. Orphanet Journal of Rare Diseases (2015) 10:15 Page 8 of 10

(See figure on previous page.) Figure 3 Genetics of the German DFNA67 family. A Graphical view of the LOD score calculation of genome-wide SNP mapping. A 3.4 Mb region on chromosome 12 and an 8.4 Mb region on chromosome 20 showed potential linkage with the phenotype. B Ideogram of chromosome 20 with the position of OSBPL2 indicated (red bar). Schematic representation of the mapped sequencing reads (forward strand) visualized with the Integrative Genomics Viewer (IGV) for patient IV:10. The c.141_142delTG (p.Arg50Alafs*103) mutation in OSBPL2 was present in half of the reads covering this region of the gene. C Electropherogram of a heterozygous carrier of the OSBPL2 mutation in exon 3 (deleted nucleotides are boxed). The localization of the mutation is indicated in a scheme of the OSBPL2 gene. D RT-PCR demonstrates Osbpl2 expression at the transcriptional level in mouse (lane 2) and rat (lane3) cochlea. Lane 1, no cDNA as negative control.

(wild-type: 480 residues) including 102 unrelated amino It remains to be determined if the interaction of both acids (p.Arg50Alafs*103) or in an unstable mRNA under- proteins is reflected by (at least partial) cellular co- going nonsense-mediated decay. OSBPL2 interacts with localization. diaphanous homologue 1 (DIAPH1) [42], the gene mu- Of note, an OSBPL2 frameshift mutation in close tated in human ADNSHL type 1 (DFNA1) [43]. DIAPH1 proximity to the nucleotide position affected by the is a Rho effector protein that regulates cytoskeletal dy- mutation reported herein has recently been described to namics by interacting with actin, microtubules and other co-segregate with ADNSHL in a large Chinese family proteins associated with cytoskeleton function [44,45]. [49]. Similar to the German DFNA67 family, age of on- Mutations in DIAPH1 are thought to impair the structural set was variable (5 to 32 years), and hearing loss was integrity of hair cells’ stereocilia, which strongly depends progressive, ranging from mild to profound. This add- on their actin cytoskeleton, and of the kinocilium, which itional DFNA67 family strongly supports the association is built around a microtubular backbone. As is assumed of OSBPL2 mutations with ADNSHL. for DIAPH1, OSBPL2 could play a role for the mainten- A truncating variant in a gene closely neighboring the ance of hair cells’ cytoskeleton, which would be compat- chromosome 20 candidate locus, a nonsense mutation in ible with the prominent presence of OSBPL2 protein at SLC17A9, p.Ser96*, is unlikely to cause hearing loss stereocilia (Figure 4). OSBPL2 binds phosphatidylinositol because it was present in healthy individuals of both our (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) [46], a phospholipid family and our in-house database of 511 epilepsy of the plasma membrane that is crucial for defining neur- exomes, and in the general population. SLC17A9 en- onal polarity [47,48], a possible hint that OSBPL2 could codes a vesicular nucleotide transporter [50], and het- be needed to establish and maintain polarity of hair cells. erozygous mutations in this gene have recently been

Figure 4 OSBPL2 expression in stereocilia of mouse cochlear inner and outer hair cells. A, B OSBPL2 (green) is expressed in stereocilia of cochlear outer hair cells (OHC) as demonstrated by co-immunostaining with anti-prestin antibody (red) of mature (P20) mice. Upon omission of the primary antibody no immunostaining can be seen, which demonstrates the specificity of the OSBPL2 antibody (inset). C, D OSBPL2 (green) is also expressed in stereocilia of cochlear inner hair cells (IHC) as demonstrated by co-immunostaining with anti-otoferlin antibody (red). Nuclei are stained with DAPI (blue). Scale bars, 10 μm. Thoenes et al. Orphanet Journal of Rare Diseases (2015) 10:15 Page 9 of 10

reported to cause disseminated superficial actinic poro- Hannover Medical School, Hannover, Germany. 7Department of Voice, Speech keratosis, DSAP [51]. None of the 12 individuals from and Hearing Disorders, University Medical Center Hamburg-Eppendorf, Hamburg, Germany. 8Department of Human Genetics, University Medical Center our DFNA67 family who carried the p.Ser96*SLC17A9 mu- Hamburg-Eppendorf, Hamburg, Germany. 9Center for Human Genetics, tation had any skin abnormalities. In conclusion, Bioscientia, Ingelheim, Germany. 10Renal Division, Department of Medicine, 11 SLC17A9 is not only unrelated to hearing loss in this University Medical Center Freiburg, Freiburg, Germany. Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), family; its haploinsufficiency does not seem to cause University of Cologne, Cologne, Germany. DSAP either. 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